Polymorphisms in endothelial nitric oxide synthase gene in early and late severe preeclampsia

Preeclampsia (PE) is characterized by hypertension and proteinuria, occurring after the 20th week of pregnancy in women who have had no previous symptoms. The disease progresses with generalized vasoconstriction and endothelial dysfunction. Clinically, it is important to diagnose the severe form of the disease (sPE), in which blood pressure and proteinuria are much higher. Recently, the gestational age (GA) of the onset of PE has led to the classification of this disease as early (GA <34 weeks) and late (GA >= 34 weeks). Several genetic polymorphisms affecting endothelial nitric oxide synthase (eNOS) levels or function were described, including G894T (Glu298Asp), VNTR b/a (variable-number 27-bp tandem repeat) and T-786C (promoter) polymorphisms. Thus, the aim of this study was to compare the distribution of G894T, VNTR b/a and T-786C polymorphisms and their haplotypes in Brazilian early and late sPE, as well as in normotensive pregnant. A total of 201 women were evaluated, 53 with early sPE, 45 with late sPE and 103 as normotensive pregnant women. The frequency of 894T allele was higher in late sPE vs normotensive pregnant, and 894TT genotype was higher in late sPE vs early sPE and normotensive pregnant. For VNTR b/a polymorphism, higher frequencies of aa genotype and a allele were observed in early sPE vs late sPE and normotensive pregnant. Besides, the frequency of haplotype T-b-C was higher in late sPE vs early sPE and normotensive pregnant. Considering the results found for eNOS polymorphisms, it is possible to suggest that the functional alterations induced by these two polymorphisms may influence the time of severe PE onset, although both alterations are putatively associated with low NO bioavailability. However, other studies are necessary to validate these findings and clarify this issue. (C) 2014 Elsevier Inc. All rights reserved.

Evidence of the Importance of the First Intracellular Loop of Prokineticin Receptor 2 in Receptor Function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2. (Molecular Endocrinology 26: 1417-1427, 2012)

Endogenous testosterone increases leukocyte-endothelial cell interaction in spontaneously hypertensive rats

Aims: Inflammation may have an important role in the beginning and in the progress of cardiovascular diseases. Testosterone exerts important effects on vascular function, which is altered in arterial hypertension. Thus, the aim of this study was to evaluate the influence of endogenous testosterone on leukocyte behavior in post-capillary venules of the mesenteric bed of spontaneously hypertensive rats (SHR). Main methods: 18 week-old intact SHR, castrated SHR and normotensive rats (intact Wistar) were used. Blood pressure was measured by tail plethysmography and serum testosterone levels by ELISA. Leukocyte rolling, adhesion and migration were evaluated in vivo in situ by intravital microscopy. Key findings: Castration significantly reduced blood pressure and reversed the increased leukocyte rolling and adhesion observed in SHRs. Leukocyte counts and other hemodynamic parameters did not differ among groups. SHRs displayed increased protein expression of P-selectin and ICAM-1 in mesenteric venules when compared to intact Wistar. Castration of SHRs restored the protein expression of the cell adhesion molecules. Significance: The findings of the present study demonstrate the critical role of endogenous testosterone mediating the effects of hypertension increasing leukocyte-endothelial cell interaction. Increased expression of cell adhesion molecules contribute to the effects of endogenous testosterone promoting increased leukocyte rolling and adhesion in SHRs. (c) 2012 Elsevier Inc. All rights reserved.

Improvement of metabolic parameters and vascular function by metformin in obese non-diabetic rats

Aims: Metformin is an insulin sensitizing agent with beneficial effects in diabetic patients on glycemic levels and in the cardiovascular system. We examined whether the metabolic changes and the vascular dysfunction in monosodium glutamate-induced obese non-diabetic (MSG) rats might be improved by metformin. Main methods: 16 week-old MSG rats were treated with metformin for 15 days and compared with age-matched untreated MSG and non-obese non-diabetic rats (control). Blood pressure, insulin sensitivity, vascular reactivity and prostanoid release in the perfused mesenteric arteriolar bed as well as nitric oxide production and reactive oxygen species generation in isolated mesenteric arteries were analyzed. Key findings: 18-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia, insulin resistance and hyperinsulinemia. Metformin treatment improved these alterations. The norepinephrine-induced response, increased in the mesenteric arteriolar bed from MSG rats, was corrected by metformin. Indomethacin corrected the enhanced contractile response in MSG rats but did not affect metformin effects. The sensitivity to acetylcholine, reduced in MSG rats, was also corrected by metformin. Indomethacin corrected the reduced sensitivity to acetylcholine in MSG rats but did not affect metformin effects. The sensitivity to sodium nitroprusside was increased in preparations from metformin-treated rats. Metformin treatment restored both the reduced PGI2/TXA2 ratio and the increased reactive oxygen species generation in preparations from MSG rats. Significance: Metformin improved the vascular function in MSG rats through reduction in reactive oxygen species generation, modulation of membrane hyperpolarization. correction of the unbalanced prostanoids release and increase in the sensitivity of the smooth muscle to nitric oxide. (c) 2011 Elsevier Inc. All rights reserved.

Vascular endothelial growth factor A (VEGFA) modulates bovine placenta steroidogenesis in vitro

Our objectives were to investigate the possible role of VEGFA in bovine placenta steroid synthesis and to determine whether cloned derived placental cells present similar responses as non-cloned ones. Placental cells from cloned (term) and non-cloned (days 90, 150, 210 and term) pregnancies were isolated and treated with VEGFA (50 ng/ml) for 24, 48 or 96 h. Progesterone (P-4) and estrone sulfate (E1S) were assessed by RIA, while aromatase P450-positive cells were quantified using the point counting test. The percentages of steroidogenic and non-steroidogenic populations were determined by flow cytometry. VEGFA augmented or decreased P-4 and E1S concentrations as well as aromatase P450-positive cell density, depending on gestational age and time in culture. The percentage of steroidogenic cells was lower than that of non-steroidogenic ones for each culture time (P < 0.05). VEGFA treatment did not change the proportion of steroidogenic and non-steroidogenic cells. Placental cells derived from cloned pregnancies presented higher concentrations of E1S and P4 than the non-cloned group. However, aromatase P450-positive cells were similar between groups (P > 0.05). VEGFA treatment altered P-4 and E1S levels in placental cells depending on type of gestation. These results suggest that VEGFA acts locally in the bovine placenta to modulate steroidogenesis during gestation, but in a different pattern between cloned and non-cloned derived placental cells at term. Therefore, this factor can be considered an important regulator of placental development and function. (C) 2012 Elsevier Ltd. All rights reserved.

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase-2 (MMP-2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. MMP-2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol-treated rats showed increased activity of MMP-2. Histopathological investigation of kidneys from ethanol-treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol-treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS-derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol-treated rats might contribute to progressive renal damage.

Phosphorus Is Associated with Coronary Artery Disease in Patients with Preserved Renal Function

High serum phosphorus levels have been associated with mortality and cardiovascular events in patients with chronic kidney disease and in the general population. In addition, high phosphorus levels have been shown to induce vascular calcification and endothelial dysfunction in vitro. The aim of this study was to evaluate the relation of phosphorus and coronary calcification and atherosclerosis in the setting of normal renal function. This was a cross-sectional study involving 290 patients with suspected coronary artery disease and undergoing elective coronary angiography, with a creatinine clearance >60 ml/min/1.73 m(2). Coronary artery obstruction was assessed by the Friesinger score and coronary artery calcification by multislice computed tomography. Serum phosphorus was higher in patients with an Agatston score >10 than in those with an Agatston score <= 10 (3.63 +/- 0.55 versus 3.49 +/- 0.52 mg/dl; p = 0.02). In the patients with Friesinger scores >4, serum phosphorus was higher (3.6 +/- 0.5 versus 3.5 +/- 0.6 mg/dl, p = 0.04) and median intact fibroblast growth factor 23 was lower (40.3 pg/ml versus 45.7 pg/ml, p = 0.01). Each 0.1-mg/dl higher serum phosphate was associated with a 7.4% higher odds of having a Friesinger score >4 (p = 0.03) and a 6.1% greater risk of having an Agatston score >10 (p = 0.01). Fibroblast growth factor 23 was a negative predictor of Friesinger score ( p = 0.002). In conclusion, phosphorus is positively associated with coronary artery calcification and obstruction in patients with suspected coronary artery disease and preserved renal function.

Mechanisms of endothelial dysfunction in obesity-associated hypertension

Obesity is strongly associated with high blood pressure, dyslipidemia, and type 2 diabetes. These conditions synergistically increase the risk of cardiovascular events. A number of central and peripheral abnormalities can explain the development or maintenance of high blood pressure in obesity. Of great interest is endothelial dysfunction, considered to be a primary risk factor in the development of hypertension. Additional mechanisms also related to endothelial dysfunction have been proposed to mediate the development of hypertension in obese individuals. These include: increase in both peripheral vasoconstriction and renal tubular sodium reabsorption, increased sympathetic activity and overactivation of both the renin-angiotensin system and the endocannabinoid system and insulin resistance. The discovery of new mechanisms regulating metabolic and vascular function and a better understanding of how vascular function can be influenced by these systems would facilitate the development of new therapies for treatment of obesity-associated hypertension.

Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

The role of NPxY domains in LRP1 receptor maturation and function

Analyses of low density lipoprotein receptor-related protein 1 (LRP1) mutant mouse embryonic fibroblasts (MEFs) generated from LRP1 knock-in mice revealed that inefficient maturation and premature proteasomal degradation of immature LRP1 is causing early embryonic lethality in NPxY1 and NPxY1+2 mutant mice. In MEFs, NPxY2 mutant LRP1 showed efficient maturation but, as expected, decreased endocytosis. The single proximal NPxY1 and the double mutant NPxY1+2 were unable to reach the cell surface as an endocytic receptor due to premature degradation. In conclusion, the proximal NPxY1 motif is essential for early sorting steps in the biosynthesis of mature LRP1.rnThe viable NPxY2 mouse was used to provide genetic evidence for LRP1-mediated amyloid-β (Aβ) transport across the blood-brain barrier (BBB). Here, we show that primary mouse brain capillary endothelial cells (pMBCECs) express functionally active LRP1. Moreover, demonstrate that LRP1 mediates [125I]-Aβ1-40 transcytosis across pMBCECs in both directions, whereas no role for LRP1-mediated Aβ degradation was detected. Aβ transport across pMBCECs generated from NPxY2 knock-in mice revealed a reduced Aβ clearance in both directions compared to WT derived pMBCECs. Finally, we conclude that LRP1 is a bona-fide receptor involved in bidirectional transcytosis of Aβ across the BBB.rn

Caveolin-1 opens endothelial cell junctions by targeting catenins

A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown.

In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

Indications for a protective function of beta2-glycoprotein I in thrombotic thrombocytopenic purpura

It has been shown that β(2) -glycoprotein I (β(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that β(2) GPI might play a role in TTP. We found that β(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that β(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of β(2) GPI to VWF by surface plasmon resonance, and determined that domain I of β(2) GPI is the binding site of VWF A1 domain. Adhesion of β(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that β(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that β(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.